Use of novel methods to study the biochemical mechanisms of ACTG2 mutations in visceral myopathy

项目摘要 这项提议的目标是研究伽马平滑肌肌动蛋白突变的机制。 (ACTG2)通过建立新开发的方法来纯化野生型和突变型，从而导致内脏肌病 ACTG2蛋白。内脏肌病是以流畅无力为特征的危及生命的疾病。 肠道、膀胱和子宫的肌肉。内脏平滑肌无力会导致腹胀， 呕吐、排尿困难、腹痛和营养不良。内脏肌病的现有治疗方法， 包括手术切除和静脉营养，是针对症状的，而不是针对目标的 疾病机制的基础。尽管目前有治疗方法，但内脏肌病患者 他们一生中的很大一部分时间都在医院，接受多项侵入性手术，往往在童年时死亡。 大约一半的内脏肌病患者存在ACTG2杂合突变。肌动蛋白是一种高度 在肌肉组织中与肌球蛋白形成收缩细丝的保守蛋白质。它们的作用机制 ACTG2突变导致内脏肌病目前尚不清楚。研究ACTG2的一个主要障碍 突变是缺乏产生纯净和具有适当翻译后翻译后的重组肌动蛋白的方法 修改。这项工作建立在最近未发表的纯化重组野生型和突变体的进展的基础上 ACTG2蛋白的体外生化特性及新纯化方法的优化 研究一组与内脏肌病相关的具有代表性的未定性ACTG2变异。研究将会 用纯化的野生型和突变型ACTG2蛋白进行多种体外实验，以表征ACTG2的作用 突变在1)肌动蛋白聚合和细丝稳定性，2)与相关的肌动蛋白结合蛋白相互作用， 在体内调节ACTG2的动力学；3)平滑肌肌球蛋白对ACTG2的作用力 细丝。此外，这项工作将确定杂合的ACTG2突变是否会产生显性的负面影响 通过与野生型和突变蛋白的混合物进行检测，对野生型ACTG2进行了鉴定。这项工作的最终目标是 确定ACTG2突变致病的生化机制。这项工作是必要的 开发以机制为基础的治疗方法，以提高患者的预期寿命和生活质量 死于内脏肌病。研究在Robert Heuckeroth博士、Roberto博士的实验室进行 多明格斯和迈克尔·奥斯塔普博士已经建立了密切的合作来研究ACTG2。多明格斯博士 是肌动蛋白结构和生物化学专家。奥斯塔普博士是肌球蛋白与肌动蛋白相互作用的专家。Dr。 Heuckeroth是包括内脏肌病在内的肠道动力障碍方面的专家，并照顾患有 ACTG2突变。这项工作通过对培养的平滑细胞中这些相同的ACTG2突变的平行研究而得到加强 肌肉细胞，转化为平滑肌的干细胞，以及(很快)ACTG2突变小鼠模型。许多 宾夕法尼亚肌肉学院的合作者还促进了基础广泛的机械性训练，这是 年轻的内科医生兼科学家计划成为一名独立的调查员。

Project Summary The goal of this proposal is to study the mechanisms by which mutations in gamma smooth muscle actin (ACTG2) cause visceral myopathy by building upon newly developed methods to purify wildtype and mutant ACTG2 proteins. Visceral myopathies are life-threatening diseases characterized by weakness of smooth muscle in the bowel, bladder and uterus. Visceral smooth muscle weakness causes abdominal distension, vomiting, urination difficulties, abdominal pain and malnutrition. Current treatments of visceral myopathy, including surgical bowel resection and intravenous nutrition, are directed at symptoms and do not target underling disease mechanisms. Despite current treatments, people suffering from visceral myopathy are hospitalized for a significant portion of their life, undergo multiple invasive surgeries and often die in childhood. Approximately half of patients with visceral myopathy have heterozygous mutations in ACTG2. Actin is a highly conserved protein that forms contractile filaments with myosin in muscle tissue. The mechanisms by which ACTG2 mutations cause visceral myopathy are currently unknown. One major obstacle to studying ACTG2 mutations is the lack of methods for producing recombinant actin that is pure and has proper post-translational modifications. This work builds on recent unpublished advances to purify recombinant wildtype and mutant ACTG2 proteins for in vitro biochemical characterization and involves optimization of novel purification methods to study a representative set of uncharacterized ACTG2 variants associated with visceral myopathy. Studies will use purified wildtype and mutant ACTG2 proteins in multiple in vitro assays to characterize the effects of ACTG2 mutations on 1) actin polymerization and filament stability, 2) interaction with relevant actin-binding proteins that regulate ACTG2 dynamics in vivo, and 3) the ability of smooth muscle myosin to generate force on ACTG2 filaments. In addition, this work will determine if heterozygous ACTG2 mutations exert dominant negative effects on wildtype ACTG2 via assays with mixtures of wildtype and mutant proteins. The ultimate goal of this work is to determine the biochemical mechanisms through which ACTG2 mutations cause disease. This work is needed to develop mechanism-based treatments to improve the life expectancy and quality of life for individuals suffering from visceral myopathy. Studies are performed in the laboratories of Dr. Robert Heuckeroth, Dr. Roberto Dominguez and Dr. Michael Ostap who have established a close collaboration to study ACTG2. Dr. Dominguez is an actin structure and biochemistry expert. Dr. Ostap is an expert on myosin interactions with actin. Dr. Heuckeroth is an expert on bowel motility disorders including visceral myopathy and cares for children with ACTG2 mutations. This work is enhanced by parallel studies of these same ACTG2 mutations in cultured smooth muscle cells, stem cells converted to smooth muscle, and (soon) an ACTG2 mutant mouse model. Many Pennsylvania Muscle Institute collaborators also facilitate broad-based, mechanistic training that is ideal for a young physician-scientist planning to become an independent investigator.